Immunoturbidimetric assay reagents and assay kits

Abstract

To provide a reagent for immunonephelometry that can accurately measure an object to be measured in a sample and a kit therefor.SOLUTION: The present invention discloses a reagent for immunonephelometry that comprises a carrier particle that is sensitized by a specific binding substance to an object for measuring, and casein or a salt thereof. There is also provided a kit therefor.SELECTED DRAWING: None

Description

[Technical field]

[0001] The present invention relates to a reagent and a kit for immunoturbidimetric measurement of an object to be measured in a test sample. [Background technology]

[0002] Many immunological measurement methods utilizing the specific binding reaction between antigens and antibodies are known, including enzyme immunoassay, fluorescent enzyme immunoassay, immunoturbidimetric assay, immunonephelometric assay, and immunochromatography.

[0003] In particular, the immunoturbidimetric method is widely used because it is easy to operate and can be automated. The immunoturbidimetric method detects the target substance by reacting carrier particles sensitized with specific antibodies with the target substance and measuring the absorbance of the complex aggregates formed by the specific binding reaction.

[0004] However, immunoassay methods have a problem in that they cannot accurately measure the object to be measured due to non-specific reactions. In order to prevent non-specific reactions, immunoassay reagents containing various proteins, surfactants, salts, etc. have been proposed. For example, there is a method using casein of 900,000 daltons or more to prevent non-specific reactions of labeled ligands (Patent Document 1), and a method using a non-specific reaction inhibitor consisting of proteins, polymers, salts, or a combination of these (Patent Document 2). [Prior art documents] [Patent documents]

[0005] [Patent Document 1] JP 2004-117341 A [Patent Document 2] JP 2010-127827 A Summary of the Invention [Problem to be solved by the invention]

[0006] An object of the present invention is to provide an immunoturbidimetric measurement reagent and a measurement kit that can accurately measure an object to be measured in a test sample. [Means for solving the problem]

[0007] As a result of extensive research into solving the above-mentioned problems, the inventors discovered that the above-mentioned problems can be achieved by a reagent and a measurement kit for immunoturbidimetric measurement of an object to be measured, which comprises carrier particles sensitized with a specific binding substance for the object to be measured, and casein or a salt thereof, and thus completed the present invention.

[0008] The present invention comprises the following. 1. A reagent for immunoturbidimetric measurement of an object to be measured, comprising carrier particles sensitized with a substance that specifically binds to the object to be measured, and casein or a salt thereof. 2. The immunoturbidimetric measurement reagent according to the preceding item 1, wherein the specific binding substance is an antibody or an antigen that specifically binds to the object to be measured. 3. The immunoturbidimetric assay reagent according to item 1 or 2 above, wherein the carrier particles are any one of cellulose particles, latex particles, metal nanoparticles, and gelatin particles. 4. The immunoturbidimetric measurement reagent according to any one of items 1 to 3 above, wherein when the measurement reagent is in liquid form, the carrier particles are contained at 0.10 to 0.25 w / v % and the casein or a salt thereof is contained at 0.10 to 1.00 w / v %. 5. The reagent for immunoturbidimetric measurement according to any one of the preceding items 1 to 4, further comprising a saccharide and / or a surfactant. 6. The immunoturbidimetric measurement reagent according to any one of items 1 to 5 above, wherein the object to be measured is C-reactive protein. 7. A kit for measuring a measurement target by an immunoturbidimetric method, characterized in that the immunoturbidimetric measurement reagent described in any one of the preceding items 1 to 6 is applied to a measurement device, and the measurement reagent is kept in a dry state until it is used for measurement. 8. A kit for measuring an object to be measured by an immunoturbidimetric method, characterized in that a reagent for measuring the object to be measured by an immunoturbidimetric method, which comprises carrier particles sensitized with a specific binding substance for the object to be measured and casein or a salt thereof, is applied to a measurement device, and the measurement reagent is kept in a dry state until it is used for measurement. 9. A method for measuring an object to be measured by mixing a test sample with a measurement reagent applied to the measurement kit described in the preceding paragraph 7 or 8, and measuring a complex aggregate produced by a specific binding reaction between the carrier particles contained in the measurement reagent and the object to be measured in the test sample. 10. A method for producing a kit for measuring an object to be measured by immunoturbidimetry, comprising the steps of: 1) applying a reagent for immunoturbidimetric measurement of the object to be measured, the reagent containing carrier particles sensitized with a substance that specifically binds to the object to be measured and casein or a salt thereof, to a measurement device; 2) A step of drying the measuring reagent applied to the measuring device. Effect of the Invention

[0009] According to the present invention, a reagent and a kit for immunoturbidimetric measurement of an object to be measured, which contain carrier particles sensitized with a specific binding substance for the object to be measured and casein or a salt thereof, non-specific reactions of the carrier particles can be efficiently suppressed, and the object to be measured in a test sample can be accurately measured. [Brief description of the drawings]

[0010] [Figure 1] FIG. 13 is a graph showing the relationship between the average concentration-converted values ​​(measured values) and CRP-free serum and CRP control samples with known CRP concentrations (0.4, 0.8, 1.6, 3.0 mg / dL) (Example 2). DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

[0011] The present invention will be described in detail below.

[0012] (1) Measurement reagents The "measurement reagent" of the present invention is characterized by containing carrier particles sensitized with a substance that specifically binds to the object to be measured, and casein or a salt thereof.

[0013] In this specification, the term "carrier particles" refers to carrier particles that can be used in measurements by immunoturbidimetry, and is not particularly limited as long as it can sensitize a specific binding substance to the measurement target. Examples of carrier particles include cellulose particles, latex particles, gold nanoparticles, gelatin particles, liposomes, microcapsules, particles such as red blood cells, etc., and cellulose particles, latex particles, gold nanoparticles, and gelatin particles are preferred, and cellulose particles are particularly preferred.

[0014] In the measurement by the immunoturbidimetric method, the average particle diameter of the carrier particles is not particularly limited. For reasons such as the ease of formation of a complex aggregate formed by a specific binding reaction between the carrier particles sensitized with a substance specifically binding to the object to be measured and the object to be measured in the test sample, and the ease of measurement of the formed complex aggregate, the average particle diameter of the carrier particles is preferably 50 nm to 1000 nm, and particularly preferably 100 nm to 400 nm. Two or more types of carrier particles having different particle diameters or shapes may be used.

[0015] In the present specification, the term "specific binding substance" is not particularly limited as long as it is a substance that causes a specific binding reaction with the object to be measured. Substances that can be used in antigen-antibody reactions are preferred, and in particular, antibodies or antigens that specifically bind to the object to be measured are preferred. When the object to be measured is an antigen, an antibody corresponding to the antigen can be used, for example, a monoclonal antibody, a polyclonal antibody, a chimeric antibody, a human antibody, or a single chain antibody (scFv), and fragments of these antibodies (Fab, F(ab')2), Fab', Fv, sFv, dsFv, etc.) can be appropriately selected and used. In addition, a derivative can be used by binding a protein or a low molecular weight compound to the antibody or antibody fragment. The origin of the antibody is not particularly limited, and examples include mammals (mouse, rabbit, rat, sheep, goat, or horse, etc.) or birds (chicken, quail, pheasant, ostrich, or duck, etc.). When the object to be measured is an antibody, it is sufficient to use an antigen for that antibody, for example, the antigen for the antibody exemplified above can be used as the antigen.

[0016] In the present invention, "casein or a salt thereof" is used as a blocking agent for suppressing non-specific reactions of carrier particles. Examples include αs-casein, β-casein, and κ-casein, and sodium hydroxide may be added to these caseins. Casein or a salt thereof is preferably Na-caseinate.

[0017] The measuring reagent of the present invention may further contain additives such as sugars and surfactants. Examples of sugars include sucrose, sorbitol, mannitol, trehalose, lactose, xylitol, galactose, allose, glucose, altrose, etc., and sucrose, sorbitol, and mannitol are particularly preferred. Examples of surfactants include amphoteric surfactants and nonionic surfactants, and examples of such surfactants include CHAPS, CHAPSO, ASB-14, C7Bz0, Tween20, Briji35, I GEPAL CA-630, and digitonin, and CHAPS and Briji35 are particularly preferred.

[0018] (2) Method for preparing measurement reagents In the measurement reagent of the present invention, the sensitization of carrier particles with a substance that specifically binds to the object to be measured is not particularly limited, but can be prepared, for example, as follows: A specific binding substance such as an antibody is added to the carrier particles, the mixture is stirred in an aqueous solvent, and the carrier particles that have been sensitized with the substance that specifically binds to the object to be measured are washed by centrifugation to prepare the carrier particles.

[0019] The measurement reagent of the present invention can be prepared by adding various aqueous solvents to the carrier particles sensitized with the binding substance, and then adding casein or a salt thereof. The above-mentioned additives and specific binding substances may be added. Examples of the aqueous solvent include purified water, physiological saline, Tris buffer, phosphate buffer, etc., and the pH of the aqueous solvent is generally, for example, pH 5 to 10. The specific binding substance can be added not only to the carrier particles but also to the measurement reagent in order to adjust the sensitivity to the specific binding reaction of the target substance in the test sample with the carrier particles sensitized with the specific binding substance for the target substance.

[0020] The liquid measurement reagent of the present invention prepared by the above method contains 0.10 to 0.50 w / v%, preferably 0.15 to 0.25 w / v%, of carrier particles sensitized with a specific binding substance for the object to be measured, and 0.05 to 5.00 w / v%, preferably 0.10 to 1.00 w / v%, of casein or a salt thereof.

[0021] (3) Measurement kit The present invention also includes a "measurement kit" for measuring an object to be measured by the immunoturbidimetric method. The kit is characterized in that the measurement reagent of the present invention is applied to a measurement device and dried, and the measurement reagent is kept in a dry state until it is used for measurement.

[0022] In the measurement kit of the present invention, the "measurement device" includes at least a sample supply part for supplying a test sample, and a reaction part to which the measurement reagent of the present invention containing carrier particles sensitized with a specific binding substance for the object to be measured is applied. It is preferable that the reaction part further includes a path for introducing the test sample supplied to the sample supply part to the reaction part as a component. The reaction part preferably has an upper part and a lower part as parts to which the measurement reagent of the present invention is applied, and may have any shape that allows the measurement reagent and the test sample to be mixed, for example, it is preferable that the reaction part is concave.

[0023] (4) Manufacturing method of the measurement kit The present invention also includes a "method of producing a measurement kit" for a measurement object by immunoturbidimetry. The method for producing the measuring kit of the present invention is characterized by comprising the steps of applying the measuring reagent of the present invention to a measuring device and drying the measuring reagent of the present invention.

[0024] In the "method for producing a measurement kit" of the present invention, it is preferable to apply the measurement reagent of the present invention to the reaction part of the measurement device, and the measurement reagent of the present invention may be applied to the measurement device in a liquid state and then dried, or the measurement reagent of the present invention in a dry state may be applied to the measurement device. Examples of the method for drying the measurement reagent of the present invention include natural drying and freeze drying.

[0025] (5)Measurement method The present invention also includes a "method for measuring an object to be measured." The method for measuring an object to be measured of the present invention is a method for measuring an object to be measured by mixing a test sample with a measurement reagent applied to the measurement kit of the present invention, and measuring a complex aggregate formed by a specific binding reaction between the object to be measured in the test sample and carrier particles sensitized with a substance that specifically binds to the object to be measured, which are contained in the measurement reagent.

[0026] The measurement method of the present invention will now be described in detail. 1) When a test sample is supplied to a sample supply section of a measurement device included in a measurement kit of the present invention, the test sample moves to a reaction section, for example, through a path in the measurement device. Since the reaction section is coated with the measurement reagent of the present invention, the test sample dissolves the measurement reagent of the present invention from a dry state, and the measurement reagent of the present invention and the test sample are mixed. The measurement reagent of the present invention may be coated only on the upper part of the reaction section, only on the lower part, or on both parts. 2) When the measurement reagent of the present invention is mixed with a test sample, a complex aggregate is generated by a specific binding reaction between the carrier particles contained in the measurement reagent of the present invention and the object to be measured in the test sample. 3) The solution containing the generated complex aggregates is irradiated with light and the absorbance is measured, whereby the substance to be measured contained in the test sample can be detected.

[0027] In the measurement method of the present invention, the temperature at which the measurement reagent of the present invention and the test sample are reacted in the reaction section is, for example, 0 to 50° C., and preferably 4 to 40° C. When irradiating the solution containing the generated complex aggregates with light, it is preferable to irradiate the reaction section with light, and the light can be irradiated, for example, with a spectrophotometer or the like.

[0028] The "test sample" to be subjected to the measurement method of the present invention may be a specimen obtained from a living body itself, or may be prepared from a specimen. For example, the test sample may be a blood sample, a body fluid, a urine sample, etc. For example, the blood sample may be whole blood, serum, plasma, etc.

[0029] In this specification, the term "measurement subject" refers to a test subject in a general clinical test, and specifically includes various components contained in blood, urine, saliva, secretions, etc., and various components contained in extracts from solids such as tissues and stool. Examples of the measurement targets include proteins such as CRP, albumin, AST, ALT, γGTP, hemoglobin, hemoglobin A1c, myoglobin, transferrin, lactoferrin, cystatin C, ferritin, α-fetoprotein, carcinoembryonic antigen, CA19-9, prostate specific antigen, fibrinogen degradation products (FDP), pepsinogen I and II, and collagen; lipid proteins such as high density lipoprotein, low density lipoprotein, and very low density lipoprotein; nucleic acids such as deoxyribonucleic acid and ribonucleic acid; enzymes such as alkaline phosphatase, lactate dehydrogenase, lipase, and amylase; immunoglobulins such as IgG, IgM, IgA, IgD, and IgE; hepatitis B virus, hepatitis C virus, human immunodeficiency virus, and Helicobacter pylori; and hormones such as sex hormones. In particular, CRP is included. CRP is a serum protein that shows a precipitation reaction with the C-polysaccharide of Streptococcus pneumoniae, and is usually present in small amounts in serum. The increase in blood CRP concentration increases rapidly when various inflammatory diseases occur. For this reason, CRP is measured as an inflammation marker to diagnose diseases such as infections and inflammation. EXAMPLES

[0030] The present invention will be described in detail below with reference to examples and comparative examples, but the present invention is not limited to these examples.

[0031] (Example 1) Evaluation of non-specific reaction inhibition of casein Na and evaluation of accuracy and repeatability In Example 1, the measurement reagent of the present invention was evaluated for inhibition of non-specific reactions of casein Na, and for accuracy and simultaneous reproducibility.

[0032] 1) Preparation of anti-CRP antibody-sensitized cellulose particles Cellulose particles were sensitized with anti-CRP antibodies as follows: Cellulose particles with an average particle size of 330 nm (NanoAct (registered trademark), sold by Asahi Kasei Fibers Corporation) were stirred in a buffer solution to a concentration of 0.1 w / v% and anti-CRP antibodies to a concentration of 100 μg / ml, and then the anti-CRP antibody-sensitized cellulose particles were washed by centrifugation to prepare the particles.

[0033] 2) Preparation of measurement reagents Purified water, anti-CRP antibody, sodium caseinate, sugars, and surfactants were added to the anti-CRP antibody-sensitized cellulose particles obtained in 1) above to prepare a measurement reagent. As a comparative example, measurement reagents were prepared by adding bovine serum albumin (BSA) or skim milk instead of sodium caseinate. The composition of each measurement reagent is summarized in Table 1.

[0034] [Table 1]

[0035] 3) Preparation of the measurement kit 0.45 μL and 0.5 μL of the measurement reagent prepared in 2) above were applied to the upper and lower parts of the reaction area of ​​the measurement device, respectively, and allowed to dry naturally at room temperature to prepare a measurement kit.

[0036] 4) Preparation of CRP control samples rCRP (sold by Oriental Yeast Co., Ltd.) was diluted with CRP-free serum (sold by Oriental Yeast Co., Ltd.) to prepare CRP control samples of 0.4 mg / dL, 0.8 mg / dL, 1.6 mg / dL, and 3.0 mg / dL. The CRP-free serum and the CRP control samples prepared above were introduced into the sample supply section of the measurement device and mixed with the measurement reagent.

[0037] 5) Measurement of absorbance CRP-free serum and CRP control specimens were introduced into the measurement device and mixed with the measurement reagent, then reacted at 37°C for 7 minutes, and the reaction area to which the measurement reagent was applied was measured at wavelengths of 630 nm (main) and 810 nm (secondary) to measure the absorbance after the reaction. CRP-free serum and CRP control specimens were each measured in triplicate.

[0038] 6) Preparation of calibration curve A calibration curve was created from the average absorbance for the CRP-free serum obtained in 4) above and CRP control samples with known CRP concentrations (0.4, 0.8, 1.6, and 3.0 mg / dL).

[0039] As a result, non-specific reactions were observed in Comparative Example 1 (BSA) and Comparative Example 2 (skim milk), and ideal calibration curves could not be obtained. In Examples 1-1 to 1-4, in which the protein added to the anti-CRP antibody-sensitized cellulose particles was sodium caseinate and the types of sugars and surfactants were changed, no non-specific reactions occurred and a concentration-dependent reaction was observed up to 0.0-3.0 mg / dL of CRP, and an ideal calibration curve could be drawn.

[0040] 7) Evaluation of accuracy and repeatability Using control sample 1 (known concentration 0.41 mg / dl) and control sample 2 (known concentration 1.04 mg / dl) as test samples, two concentrations of the control sample were measured with N=5 in the same procedure as in 5) above, and the obtained absorbance was converted into a concentration using the calibration curve (Examples 1-1 to 1-4) created above, and the accuracy and simultaneous reproducibility were evaluated.

[0041] <Accuracy assessment method> For accuracy, the average value of the two CRP control samples measured in N=5 was compared with the known concentration, and the difference in the known concentration was calculated. Note that a known concentration within ±0.2 mg / dL indicates high accuracy. <Method of evaluating repeatability> For the within-run reproducibility, the above two concentrations of CRP control samples were measured with N=5, and the standard deviation (SD) was calculated. A standard deviation of 0.1 mg / dL or less indicates high within-run reproducibility.

[0042] [Table 2]

[0043] The results of the measurement of the accuracy and on-line reproducibility of the control samples of the above two concentrations are shown in Table 2. Non-specific reactions occurred in Comparative Example 1 (BSA) and Comparative Example 2 (skim milk), and the accuracy and on-line reproducibility could not be properly evaluated. In Examples 1-1 to 1-4, in which casein Na was added, the difference in the known concentrations of the control samples of the two concentrations was within ±0.2 mg / dL, and the standard deviation was 0.1 mg / dL or less, satisfying the standards for accuracy and on-line reproducibility.

[0044] (Example 2) Confirmation of optimal concentration of sodium caseinate In Example 2, the optimal concentration of casein Na for the measurement reagent of the present invention was confirmed.

[0045] In Example 2, the measurement reagent was prepared by specifying the anti-CRP antibody-sensitized cellulose particles at 0.2 w / v%, sucrose at 0.5 w / v%, CHAPS at 0.25 w / v%, anti-CRP antibody at 100 μg / ml, and casein Na at 0.1 w / v%, 0.2 w / v%, 0.4 w / v%, 0.6 w / v%, 0.8 w / v%, and 1.00 w / v% (Table 3). The measurement reagent was prepared according to Example 1.

[0046] [Table 3]

[0047] 1) Creating a calibration curve For the measurement reagents shown in Table 3, a calibration curve was created from the average absorbance of each test sample of CRP-free serum and CRP control specimens in the same manner as in Example 1 above. A concentration-dependent reaction was observed at CRP 0.0-3.0 mg / dL for all concentrations of Na casein, and an ideal calibration curve was obtained. In addition, a graph was shown (Figure 1) showing the relationship between the average values ​​(measured values) converted from the above calibration curve and CRP-free serum and CRP control specimens with known CRP concentrations (0.4, 0.8, 1.6, 3.0 mg / dL). The average values ​​(measured values) converted from the concentrations were close to the known concentrations. [Industrial Applicability]

[0048] As described above in detail, the immunoturbidimetric measurement reagent and measurement kit of the present invention can efficiently suppress non-specific reactions, making it possible to draw an ideal calibration curve and accurately measure the substance to be measured in the test sample.

Claims

1. An immunoturbidimetric measurement device having carrier particles sensitized with a specific binding substance for the object to be measured and an immunoturbidimetric measurement reagent containing casein or a salt thereof applied thereto, the measurement device being characterized in that the immunoturbidimetric measurement reagent is kept in a dry state until it is used for measurement, a blood sample is used as a test sample, and by supplying the test sample, the object to be measured in the test sample can be measured in a concentration-dependent manner, and the device does not have a specific binding substance for the object to be measured immobilized thereon.

2. The immunoturbidimetric measurement device according to claim 1 , wherein the specific binding substance is an antibody or an antigen that specifically binds to the object to be measured.

3. The immunoturbidimetric measurement device according to claim 2 , wherein the specific binding substance is an antibody that specifically binds to the object to be measured.

4. The immunoturbidimetric measurement device according to any one of claims 1 to 3, wherein the carrier particles are any one of cellulose particles, latex particles, metal nanoparticles, and gelatin particles.

5. The immunoturbidimetric measurement device according to any one of claims 1 to 4, wherein when the immunoturbidimetric measurement reagent applied to the measurement device is in a liquid state at the time of application, the carrier particles are contained at 0.10 to 0.25 w / v % and the casein or a salt thereof is contained at 0.10 to 1.00 w / v %.

6. The immunoturbidimetric measurement device according to claim 5 , wherein the immunoturbidimetric measurement reagent applied to the measurement device further contains a sugar and / or a surfactant.

7. The immunoturbidimetric measurement device according to any one of claims 1 to 6, wherein the measurement object is C-reactive protein.

8. The immunoturbidimetric measurement device according to any one of claims 1 to 7, characterized in that the immunoturbidimetric measurement device includes, as components, a sample supply section and a reaction section to which an immunoturbidimetric measurement reagent is applied.

9. 9. An immunoturbidimetric assay kit comprising the immunoturbidimetric assay device according to claim 1, and not containing a dispersion liquid of an immunoturbidimetric assay reagent.

10. 9. A method for measuring an object to be measured, using the measurement device according to claim 8, comprising: introducing a test sample supplied from a sample supply section of the device into a reaction section to which an immunoturbidimetric measurement reagent is applied; mixing the measurement reagent with the test sample; using a blood sample as the test sample; and measuring a composite aggregate produced by a specific binding reaction between the carrier particles contained in the measurement reagent and the object to be measured in the test sample.

11. A measurement method comprising: an immunoturbidimetric measurement device coated with carrier particles sensitized with a specific binding substance for the object to be measured and an immunoturbidimetric measurement reagent containing casein or a salt thereof, wherein the immunoturbidimetric measurement reagent is kept in a dry state in the measurement device until it is used for measurement; the specific binding substance for the object to be measured is not immobilized on the device; and wherein a blood sample is used as a test sample, and by supplying the test sample, the object to be measured in the test sample can be measured in a concentration-dependent manner.

Images